Radiolabeled proteins for diagnostic or therapeutic use

ABSTRACT

Chelating compounds of specified structure are useful for radiolabeling targeting molecules such as antibodies. Cleavable linkers connect the radionuclide metal chelates to the antibodies. The radiolabeled antibodies have improved biodistribution properties, including reduced localization within the intestines and kidneys.

BACKGROUND

Radiolabeled antibodies are used in a variety of diagnostic andtherapeutic medical procedures. The increased specificity of monoclonalantibodies, compared to polyclonal antibodies, makes them even moreuseful for delivering diagnostic or therapeutic agents such asradioisotopes to desired target sites in vivo. A monoclonal antibodyspecific for a desired type of target cells such as tumor cells may beused to deliver a therapeutic radionuclide attached to the antibody tothe target cells, thereby causing the eradication of the undesiredtarget cells. Alternatively a monoclonal antibody having adiagnostically effective radionuclide attached thereto may beadministered, whereupon the radiolabeled antibody localizes on thetarget tissue. Conventional diagnostic procedures then may be used todetect the presence of the target sites within the patient.

One method for radiolabeling proteins such as antibodies involvesattachment of radionuclide metal chelates to the proteins. Chelateshaving a variety of chemical structures have been developed for thispurpose. The usefulness of such chelates is dependent upon a number offactors such as the stability of radionuclide binding within the chelateand the reactivity of the chelate with the desired protein. Theefficiency of radiolabeling of the chelating compound to produce thedesired radionuclide metal chelate also is important. Anotherconsideration is the biodistribution of the radiolabeled antibody andcatabolites thereof in vivo. Localization in non-target tissues limitsthe total dosage of a therapeutic radiolabeled antibody that can beadministered, thereby decreasing the therapeutic effect. In diagnosticprocedures, localization in non-target tissues may cause undesirablebackground and/or result in misdiagnosis. The need remains forimprovement in these and other characteristics of radionuclide metalchelate compounds used for radiolabeling of proteins such as antibodies.

SUMMARY OF THE INVENTION

The present invention provides chelating compounds, the correspondingradionuclide metal chelates, and targeting molecules such as proteinsradiolabeled therewith. The radiolabeled proteins of the presentinvention have use in various assays as well as in vivo diagnostic andtherapeutic procedures. The protein may be a monoclonal antibody thatbinds to cancer cells, for example.

Compounds of the present invention include compounds of the formulas:##STR1## wherein: m is 0 or 1;

R₁ represents H or CH₃ ;

X represents O or S;

each R₃ is independently selected from H, CH₂ OH, CH₃, and --(CH₂)_(n)--COOH, wherein n is 0 to about 2, with at least one R₃ substituentbeing --(CH₂)_(n) --COOH;

T' and T each represent hydrogen or a sulfur protecting group;

R₂ represents a spacer; and

P' represents a targeting molecule or a conjugation group; ##STR2##wherein p is 0 or 1; one of R' and R" represents ##STR3## and the otheris selected from H, CH₃, CH₂ OH, (CH₂)_(n) --CONH₂ and (CH₂)_(n) --COOHwherein n is 0 to about 2;

m is 0 or 1;

R₁ represents H or CH₃ ;

X represents O or S;

T represents hydrogen or a sulfur protecting group;

R₂ represents a spacer; and

P' represents a targeting molecule or a conjugation group; and ##STR4##wherein p is 0 or 1; each R₄ is independently selected from H, CH₃, CH₂OH, (CH₂)_(n) --CONH₂ and (CH₂)_(n) --COOH wherein n is 0 to about 2;

m is 0 or 1;

R represents H or CH₃ ;

X represents O or S;

T represents hydrogen or a sulfur protecting group;

R₂ represents a spacer; and

P' represents a targeting molecule or a conjugation group.

Reduction of undesirable localization of radioactivity within thekidneys and/or intestines has been achieved using the radiolabeledtargeting molecules of the present invention. While not wishing to bebound by theory, the improved biodistribution properties are believed tobe at least in part attributable to the presence and orientation of acleavable ester within the linkage joining the chelate to the targetingmolecule. The chemical structure of the chelating compounds and theresultant stereochemistry of the chelates released by cleavage of thelinker also may play a role in reducing localization of radioactivity inthe kidneys and intestines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-10 depict chemical synthesis procedures that may be used toprepare certain chelating compounds of the present invention.

FIG. 1 depicts a flow chart illustrating a procedure for the synthesisof serine succinate reagent.

FIG. 2 depicts a flow chart illustrating a procedure for the synthesisof cysteine-serine succinate ligand.

FIG. 3 depicts a flow chart illustrating a procedure for the synthesisof N₃ S serine succinate ligand.

FIG. 4 depicts a flow chart illustrating a procedure for the synthesisofN-(S-isobutyrylmercaptoacetyl)-O-(3-carbo-N'-hydroxysuccinimidylpropanoyl)-serylgylcylgylcine.

FIG. 5 depicts a flow chart illustrating a procedure for the synthesisof N₃ S serine succinate ligand.

FIG. 6 depicts a flow chart illustrating a procedure for the preparationof radiolabeled protein.

FIGS. 7-9 depict a flow chart illustrating a procedure for the synthesisof N₃ S serine succinate ligand.

FIG. 10 depicts a flow chart illustrating a procedure for the synthesisof a precursor to the cysteine-serine succinate ligand of FIG. 2.

FIGS. 11-15 depict biodistribution data for antibodies radiolabeled withvarious radionuclide metal chelates.

In the figures, "ID" represents injected dose, "BL" represents blood,"LV" or blood, "LI" represents "IN" represents intestines.

FIG. 11 graphically illustrates biodistribution of ^(99m) Tc-labeledantibody fragments analyzed in a rat model. The top graph representsbiodistribution data for an antibody fragment labeled with a ^(99m)Tc--N₃ S chelate wherein there is no ester linkage between the chelateand the antibody. The bottom graph represents biodistribution data forthe same antibody fragment labeled with a ^(99m) Tc-chelate preparedfrom compound 13 of FIG. 2.

FIG. 12 graphically illustrates biodistribution of ^(99m) Tc-labeledantibody fragments analyzed in a rat model. The top graph representsbiodistribution data for an antibody fragment labeled with a ^(99m)Tc-N₃ S chelate wherein there is no ester linkage between the chelateand the antibody. The bottom graph represents biodistribution data forthe same antibody fragment labeled with a ^(99m) Tc-chelate preparedfrom compound 20 of FIG. 3.

FIG. 13 graphically illustrates biodistribution of ^(99m) Tc-labeledantibody fragments analyzed in a rat model. The top graph representsbiodistribution data for an antibody fragment labeled with a ^(99m)Tc-N₃ S chelate wherein there is no ester linkage between the chelateand the antibody. The bottom graph represents biodistribution data forthe same antibody fragment labeled with a ^(99m) Tc-chelate preparedfrom compound 47 of FIG. 9.

FIG. 14 graphically illustrates biodistribution of ^(99m) Tc-labeledantibody fragments analyzed in a rat model. The top graph representsbiodistribution data for an antibody fragment labeled with a ^(99m)Tc-N₃ S chelate wherein there is no ester linkage between the chelateand the antibody. The bottom graph represents biodistribution data forthe same antibody fragment labeled with a ^(99m) Tc-N₃ S chelate whereinan ester is present in the linkage between the chelate and the antibody,but the ester has an orientation opposite to that of the compounds of hepresent invention.

FIG. 15 graphically illustrates biodistributon of ^(99m) Tc-labeledantibody fragments analyzed in a rat model. The graph represents acomposite of biodistribution data for an antibody fragment labeled witha ^(99m) Tc-N₃ S chelate wherein there is no ester linkage between thechelate and the antibody, and biodistribution data for the same antibodyfragment labeled with a ^(99m) Tc-N₃ S chelate wherein an ester ispresent in the linkage between the chelate and the antibody, but theester has an orientation opposite to that of the compounds of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides chelating compounds and radionuclidemetal chelate compounds prepared therefrom, as well as radiolabeledtargeting molecules having the chelates attached thereto. Theradionuclide metal chelates of the present invention are attached totargeting molecules such as antibodies to form radiolabeled targetingmolecules having diagnostic or therapeutic use. The compounds are boundto a targeting molecule or contain a conjugation group for attachment ofthe compound to a desired targeting molecule.

Provided by the present invention is a chelating compound of theformula: ##STR5## wherein: m is 0 or 1;

R₁ represents H or CH₃ ;

X represents O or S;

each R₃ is independently selected from H, CH₃, CH₂ OH, and (CH₂)_(n)--COOH, wherein n is 0 to about 2, with at least one R₃ substituentbeing --(CH₂)_(n) --COOH;

T' and T each represent hydrogen or a sulfur protecting group;

R₂ represents a spacer; and

P' represents a targeting molecule or a conjugation group.

R₂ may represent any suitable spacer, generally comprising at least onecarbon atom. The spacer should permit both cleaving of the ester (i.e.,##STR6## ) and reaction of a conjugation group P' with a targetingmolecule. For example, the R₂ spacer should not have a structure thatsubstantially inhibits ester cleavage or the conjugation reaction, e.g.,through steric hindrance thereof. Among the suitable R₂ spacers for thechelating compounds of the present invention, including those describedbelow, are the following:

--(CH₂)_(n') -- wherein n' is 2-5, preferably 2; ##STR7## wherein Wrepresents an electron withdrawing group (e.g., a carboxylic acid, SO₃ ⁻or nitro group) or an electron donating group (e.g., a methoxy orhydroxy group) and m' is 1-4; ##STR8## wherein W represents an optionalelectron donating or withdrawing group.

In one embodiment of the present invention, X is O and R₂ is --(CH₂)₂--. An example of such a chelating compound is the following: ##STR9##wherein the symbols are as defined above. In one embodiment of thepresent invention, T represents a hemithioacetal sulfur protecting groupsuch as an ethoxyethyl group, and T' represents an acetamidomethylsulfur protecting group. These sulfur protecting groups are describedbelow.

Another chelating compound of the present invention is of the formula:##STR10## wherein p is 0 or 1; one of R' and R" represents ##STR11## andthe other is selected from H, CH₃, CH₂ OH, (CH₂)_(n) --CONH₂ and--(CH₂)_(n) --COOH wherein n is 0 to about 2;

m is 0 or 1;

R₁ represents H or CH₃ ;

X represents O or S;

T represents hydrogen or a sulfur protecting group;

R₂ represents a spacer; and

P' represents a targeting molecule or a conjugation group.

Other substituents that may be used in position R' or R" (whicheverposition is not occupied with the ester-containing linkage terminatingin P') are the polar groups --SO₃ ⁻, --OSO₃ ⁻ --N⁺ (CH₃)₂, and the like,to further enhance water solubility of the compound.

In one embodiment of the present invention, p is 0, X is O, and R₂ is--(CH₂)₂ --. Examples of such chelating compounds are the following:##STR12## wherein the symbols are as defined above. Another chelatingcompound of the present invention is of the formula: ##STR13## wherein pis 0 or 1; each R₄ is independently selected from H, CH₃, CH₂ OH,(CH₂)_(n) --CONH₂ and --(CH₂)_(n) --COOH wherein n is 0 to about 2;

m is 0 to about 4, preferably 0 or 1;

R represents H or CH₃ ;

X represents O or S;

T represents hydrogen or a sulfur protecting group;

R₂ represents a spacer; and

P' represents a targeting molecule or a conjugation group.

Other substituents that may be used in an R₄ position are the polargroups --SO₃ ⁻, --OSO₃ ⁻, --N⁺ (CH₃)₂, and the like, to further enhancewater solubility of the compound.

In one embodiment of the present invention, each R₄ is H, p is 0, X isO, and R₂ is --(CH₂)₂ --. An example of such a chelating compound is thefollowing: ##STR14## wherein the symbols are as defined above.

The corresponding radionuclide metal chelates are represented by thefollowing formulas, respectively: ##STR15## wherein M represents aradionuclide metal or oxide thereof, and the other symbols are asdefined above. Stereochemical isomers of these chelates (which formduring the radiolabeling reaction) also are encompassed by the presentinvention.

A conjugation group is a chemically reactive functional group that willreact with a targeting molecule to bind the compound thereto. When thetargeting molecule is a protein, the conjugation group is reactive underconditions that do not denature or otherwise adversely affect theprotein. The conjugation group therefore is sufficiently reactive with afunctional group on a protein so that the reaction can be conducted in asubstantially aqueous solution and does not have to be forced, e.g. byheating to high temperatures, which may denature the protein. Examplesof suitable conjugation groups include but are not limited to activeesters, isothiocyanates, amines, hydrazines, maleimides or otherMichael-type acceptors, thiols, and activated halides. Among thepreferred active esters are N-hydroxysuccinimidyl ester,sulfosuccinimidyl ester, thiophenyl ester, 2,3,5,6-tetrafluorophenylester, and 2,3,5,6-tetrafluorothiophenyl ester. The latter threepreferred active esters may comprise a group that enhances watersolubility, at the para (i.e., 4) or the ortho position on the phenylring. Examples of such groups are CO₂ H, SO₃.sup. -, PO₃ ²⁻, OPO₃ ²⁻,OSO₃ ⁻, N⁺ R₃ wherein each R represents H or an alkyl group, and O(CH₂CH₂ O)_(n) CH₃ groups.

The targeting molecule is any molecule that will serve to deliver theradionuclide metal chelate to a desired target site (e.g., target cells)in vitro or in vivo. Examples of targeting molecules include, but arenot limited to, steroids, cholesterol, lymphokines, and those drugs andproteins that bind to a desired target site.

The targeting molecule may be a targeting protein, which is capable ofbinding to a desired target site. The term "protein" as used hereinincludes proteins, polypeptides, and fragments thereof. The targetingprotein may bind to a receptor, substrate, antigenic determinant, orother binding site on a target cell or other target site. The targetingprotein serves to deliver the radionuclide attached thereto to a desiredtarget site in vivo. Examples of targeting proteins include, but are notlimited to, antibodies and antibody fragments, hormones, fibrinolyticenzymes, and biologic response modifiers. In addition, other moleculesthat localize in a desired target site in vivo, although not strictlyproteins, are included within the definition of the term "targetingproteins" as used herein. For example, certain carbohydrates orglycoproteins may be used in the present invention. The proteins may bemodified, e.g., to produce variants and fragments thereof, as long asthe desired biological property (i.e., the ability to bind to the targetsite) is retained. The proteins may be modified by using various geneticengineering or protein engineering techniques.

Among the preferred targeting proteins are antibodies, most preferablymonoclonal antibodies. A number of monoclonal antibodies that bind to aspecific type of cell have been developed, including monoclonalanti-bodies specific for tumor-associated antigens in humans. Among themany such monoclonal antibodies that may be used are anti-TAC, or otherinterleukin-2 receptor antibodies; 9.2.27 and NR-ML-05, reactive withthe 250 kilodalton human melanoma-associated proteoglycan; and NR-LU-10,reactive with a pancarcinoma glycoprotein. The antibody employed in thepresent invention may be an intact (whole) molecule, a fragment thereof,or a functional equivalent thereof. Examples of antibody fragments areF(ab')₂, Fab', Fab, and F_(v) fragments, which may be produced byconventional methods o by genetic or protein engineering.

Targeting molecules are rarely completely specific for a desired targetsite. Localization in non-target tissues may occur throughcross-reactivity or non-specific uptake, for example. In the case ofradiolabeled targeting molecules, such localization at non-target sitesmay result in decreased clarity of diagnostic images (due to theincreased "background") and misdiagnosis. Exposure of non-target tissuesto radiation also occurs, which is especially undesirable in therapeuticprocedures. The improved biodistribution properties of the radiolabeledtargeting molecules of the present invention help to alleviate theseproblems.

The chelating compounds of the present invention comprise either twonitrogen and two sulfur donor atoms or one sulfur and three nitrogendonor atoms, and thus may be termed "N₂ S₂ " or "N₃ S" chelatingcompounds, respectively. The radiolabeled targeting proteins of thepresent invention exhibit certain improved biodistribution propertiescompared to targeting proteins radiolabeled with certain other N₂ S₂ orN₃ S chelates. Most notably, localization of radioactivity within theintestines and kidneys is reduced.

Targeting proteins radiolabeled with certain N₂ S₂ radionuclide metalchelates are described, for example, in European Patent ApplicationPublication Number 188,256 as well as co-pending U.S. patentapplications Ser. Nos. 07/065,011 and 07/367,502. The use of N₃ Schelates to radiolabel proteins is described in European patentapplication publication number 284,071. When the radiolabeled proteinsdisclosed in these previous patent applications are administered invivo, a percentage of the injected dosage of the radionuclide becomeslocalized within the intestines (i.e., becomes part of the intestinalcontents, rather than binding to intestinal epithelial tissue per se)and/or the kidneys. Although stable attachment of radionuclides toantibodies and effective localization thereof on target tumors has beenachieved using these previous protein radiolabeling systems, reductionof the localization of radioactivity in non-target organs would be adesired improvement. U.S. patent application Ser. No. 07/367,502embodies one approach for reducing non-target organ localization ofradioactivity, but further mitigation of this problem would bebeneficial.

In these earlier approaches, a portion of the non-targetboundadministered radiolabeled proteins (e.g., antibodies or fragmentsthereof) most likely is first metabolized to produce radiolabeledcatabolites that subsequently enter the intestines, probably throughhepatobiliary excretion. When the chelate is attached to lysine residuesof the targeting protein, a major catabolite appears to be the lysineadduct of the chelate. Intestinal localization of radioactivity may beconfused with (or obstruct) target sites in the abdominal area. Fortherapeutic procedures, the dosage that can be safely administered isreduced when intestinal localization occurs (due to exposure of normaltissues to the radiation). The therapeutic effect on the target sitestherefore also is reduced.

As illustrated in the examples below, the biodistribution patterns invivo differ when targeting proteins (e.g., antibody fragments) areradiolabeled with a chelate of the present invention, compared toradiolabeling using certain other N₃ S and N₂ S₂ chelates. The advantageof reduced intestinal and kidney localization is demonstrated for theradiolabeled targeting proteins of the present invention. While notwishing to be bound by theory, it is believed that the improvedbiodistribution properties of the radiolabeled proteins of the inventionare at least in part attributable to the presence and orientation of acleavable ester within the linkage joining the chelate to the protein.The chemical structure of the chelating compounds and the resultingstereochemistry of the chelates released by cleavage of the linker alsomay play a role in reducing localization of radioactivity in theintestines and kidneys.

The ester component of the linkage between the chelate core and theprotein is cleavable at the position indicated by the arrow: ##STR16##Cleaving of the ester in vivo is believed to occur due to hydrolysis,chemical elimination, and/or enzymatic cleavage (e.g., by liver orkidney esterases), or a combination thereof. However, the conjugates ofthe present invention have demonstrated sufficient stability in serumfor in vivo use.

After cleavage of the ester, the portion of the linkage still attachedto the chelate terminates in a hydroxyl group (when X is O) or asulfhydryl group (when X is S). The orientation of the ester ##STR17##also appears to be beneficial for achieving the desired biodistributionproperties. The relatively small size of the chelate released by estercleavage (compared, for example, to the abovedescribed lysine adductcatabolites) may also contribute to the efficient clearance ofradioactivity from the body. Lysine adducts of the chelates may be aminor metabolite of the radiolabeled proteins of the present invention.

The carboxylic acid substituent(s) on the compounds of the presentinvention increase the polarity, and therefore the water solubility, ofthe compounds. The increased water solubility is believed to furthercontribute to decreased hepatobiliary uptake of the radiolabeledproteins or catabolites thereof. Other substituents that enhancepolarity (e.g., a sulphonate group) may be used on the chelatingcompounds, in addition to (or instead of) the COOH substituents. Thefree carboxylic acid substituent(s) also may assist in the chelation ofthe radionuclide, thereby promoting good radiolabeling yields.

The chelating compounds of the present invention may be synthesizedusing amino acid derivatives. The various compounds within the scope ofthe present invention may be prepared by choosing amino acids having thedesired side chains to produce the different R₃, R₄ and othersubstituents, and other structural variations. For example, when R₁ ishydrogen, m is 0, and X is O, the cleavable ester is within a serinederivative. (The hydroxyl of serine is believed to be generated uponcleavage of the ester.) When R₁ is --CH₃, m is 0, and X is O, thelinkage is a threonine derivative. When R₁ is hydrogen, m is 0, and X isS, the linkage is a cysteine derivative. When m is 1, the linkage may beconsidered to be a homo derivative of the amino acids (homoserine,homothreorine, and homocysteine, respectively.)

The use of natural amino acids may contribute to recognition of theester-containing linkage by esterases in vivo. Amino acids having sidechains that enhance polarity and therefore water solubility (e.g.,--COOH and --CH₂ OH) also are generally desirable, and may be used tovary the R₃ and R₄ substituents, for example.

During radiolabeling, bonds form between the four donor atoms and theradionuclide metal to form the corresponding radionuclide metal chelate.Any suitable conventional sulfur protecting group(s) may be attached tothe sulfur donor atoms of the compounds of the present invention. Theprotecting groups should be removable, either prior to or during theradiolabeling reaction. In the case of N₂ S₂ compounds, the protectinggroups attached to the two sulfur donor atoms may be the same ordifferent. Alternatively, a single protecting group, e.g. a thioacetalgroup, may protect both sulfur donor atoms. Among the preferred sulfurprotecting groups are acetamidomethyl and hemithioacetal protectinggroups, which are displacable from the chelating compound during theradiolabeling reaction.

An acetamidomethyl sulfur-protecting group is represented by thefollowing formula, wherein the sulfur atom shown is a sulfur donor atomof the chelating compound: ##STR18##

The acetamidomethyl group is displaced from the chelating compoundduring radiolabeling conducted at about 50° C. in a reaction mixturehaving a pH of about 3 to 6.

When hemithioacetal protective groups are used, each sulfur atom to beprotected has a separate protective group attached to it, which togetherwith the sulfur atom defines a hemithioacetal group. The hemithioacetalgroups contain a carbon atom bonded directly (i.e., without anyintervening atoms) to a sulfur atom and an oxygen atom, i.e., ##STR19##

Preferred hemithioacetals generally are of the following formula,wherein the sulfur atom is a sulfur atom of the chelating compound, anda separate protecting group is attached to each of the sulfur atoms onthe chelating compound: ##STR20## wherein R³ is a lower alkyl group,preferably of from two to five carbon atoms, and R⁴ is a lower alkylgroup, preferably of from one to three carbon atoms. Alternatively, R³and R⁴ may be taken together with the carbon atom and the oxygen atomshown in the formula to define a nonaromatic ring, preferably comprisingfrom three to seven carbon atoms in addition to the carbon and oxygenatoms shown in the formula. R⁵ represents hydrogen or a lower alkylgroup wherein the alkyl group preferably is of from one to three carbonatoms. Examples of such preferred hemithioacetals include, but are notlimited to: ##STR21##

Other hemithioacetal sulfur protecting groups include those derived frommonosaccharides, such as the following, wherein the sulfur atom is asulfur donor atom of the chelating compound: ##STR22##

These sulfur-protective groups are displaced during the radiolabelingreaction, conducted at acidic pH, in what is believed to bemetal-assisted acid cleavage. Bonds form between the sulfur atoms andthe metal radionuclide. A separate step for removal of thesulfur-protective groups is not necessary. The radiolabeling procedurethus is simplified. In addition, the basic pH conditions and harshconditions associated with certain known radiolabeling procedures orprocedures for removal of other sulfur protective groups are avoided.Thus, base-sensitive groups on the chelating compound survive theradiolabeling step intact. Such base labile groups include any groupwhich may be destroyed, hydrolyzed, or otherwise adversely affected byexposure to basic pH. In general, such groups include esters,maleimides, and isothiocyanates, among others. Such groups may bepresent on the chelating compound as protein conjugation groups.

The chelating compounds of the present invention are radiolabeled, usingconventional procedures, with any of a variety of radionuclide metals toform the corresponding radionuclide metal chelates. These radionuclidemetals include, but are not limited to, copper (e.g., ⁶⁷ Cu and ⁶⁴ Cu);technetium (e.g., ^(99m) Tc); rhenium (e.g., ¹⁸⁶ Re and ¹⁸⁸ Re); lead(e.g., ²¹² Pb); bismuth (e.g, ²¹² Bi); palladium (e.g., ¹⁰⁹ Pd), andrhodium (e.g., ¹⁰⁵ Rh) Methods for preparing these isotopes are known.Molybdenum/technetium generators for producing ^(99m) Tc arecommercially available. Procedures for processing ¹⁸⁶ Re include theprocedures described by Deutsch et al., (Nucl. Med. Biol. Vol.13:4:465-477, 1986) and Vanderheyden et al. (Inorganic Chemistry. Vol.24:1666-1673, 1985), and methods for production of ¹⁸⁸ Re have beendescribed by Blachot et al. (Intl. J. of Applied Radiation and IsotopesVol. 20:467-470, 1969) and by Klofutar et al. (J. of RadioanalyticalChem., Vol. 5:3-10, 1970). Production of ¹⁰⁹ Pd is described in Fawwazet al., J. Nucl. Med. (1984), 25:796. Production of ²¹² Pb and ²¹² Bi isdescribed in Gansow et al., Amer. Chem. Soc. Symp. Ser. (1984),241:215-217, and Kozah et al., Proc. Nat'l, Acad. Sci. USA (Jan. 1986)83:474-478. ^(99m) Tc is preferred for diagnostic use, and the otherradionuclides listed above have therapeutic use.

In one embodiment of the present invention, chelating compounds of theinvention comprising acetamidomethyl and/or hemithioacetal sulfurprotective groups are radiolabeled with a metal radionuclide by reactingthe compound with the radionuclide under conditions of acidic pH. It isbelieved that the acidic pH and the presence of the metal bothcontribute to the displacement of the sulfur protective groups from thechelating compound. The radionuclide is in chelatable form when reactedwith the chelating compounds of the invention.

In the case of technetium and rhenium, being in "chelatable form"generally requires a reducing step. A reducing agent will be employed toreduce the radionuclides (e.g., in the form of pertechnetate andperrhenate, respectively) to a lower oxidation state at which chelationwill occur. Many suitable reducing agents, and the use thereof, areknown. (See, for example, U.S. Pat. Nos. 4,440,738; 4,434,151; and4,652,440.) Such reducing agents include, but are not limited to,stannous ion (e.g., in the form of stannous salts such as stannouschloride or stannous fluoride), metallic tin, ferrous ion (e.g., in theform of ferrous salts such as ferrous chloride, ferrous sulfate, orferrous ascorbate) and many others. Sodium pertechnetate (i.e., ^(99m)TcO₄ -- which is in the +7 oxidation level) or sodium perrhenate (i.e.,¹⁸⁸ ReO₄ --, ¹⁸⁶ ReO₄ --) may be combined simultaneously with a reducingagent and a chelating compound of the invention in accordance with theradiolabeling method of the invention, to form a chelate.

Preferably, the radionuclide is treated with a reducing agent and acomplexing agent to form an intermediate complex (i.e., an "exchangecomplex"). Complexing agents are compounds which bind the radionuclidemore weakly than do the chelate compounds of the invention, and may beweak chelators. Any of the suitable known complexing agents may be used,including but not limited to gluconic acid, glucoheptonic acid,methylene disphosphonate, glyceric acid, glycolic acid, tartaric acid,mannitol, oxalic acid, malonic acid, succinic acid, bicine, malic acid,N,N'-bis(2-hydroxy ethyl) ethylene diamine, citric acid, ascorbic acidand gentisic acid. Good results are obtained using gluconic acid orglucoheptonic acid as the technetium-complexing agent and citric acidfor rhenium. When the radionuclide in the form of such an exchangecomplex is reacted with the chelating compounds of the invention, theradionuclide will transfer to these compounds which bind theradionuclide more strongly to form chelates of the invention. Heating isoften required to promote transfer of the radionuclide. Radionuclides inthe form of such exchange complexes also are considered to be in"chelatable form" for the purposes of the present invention.

Chelates of ²¹² Pb, ²¹² Bi, 103Rh, and ¹⁰⁹ Pd may be prepared bycombining the appropriate salt of the radionuclide with the chelatingcompound and incubating the reaction mixture at room temperature or athigher temperatures. It is not necessary to treat the lead, bismuth,rhodium, palladium, and copper radioisotopes with a reducing agent priorto chelation, as such isotopes are already in an oxidation statesuitable for chelation (i.e., in chelatable form). The specificradiolabeling reaction conditions may vary somewhat according to theparticular radionuclide and chelating compound involved.

The chelating compound may be radiolabeled to form a radionuclide metalchelate, which then is reacted with a targeting protein. Alternatively,the unlabeled chelating compound may be attached to the targetingprotein and subsequently radiolabeled. For the second approach, sulfurprotecting group(s) that are displaceable/removable at a pH compatiblewith the presence of a protein should be used. Among the protectinggroups suitable for use in the second approach are acyl type groups suchas those of the formula ##STR23## wherein the S is a sulfur atom of thechelating compound and R is an alkyl or aryl group. Examples areS-isobutyryl, S-benzoyl, and S-acetyl protecting groups.

Proteins contain a variety of functional groups; e.g., carboxylic acid(COOH) or free amine (--NH₂) groups, which are available for reactionwith a suitable protein conjugation group on a chelator to bind thechelator to the protein. For example, an active ester on the chelatorreacts with epsilon amine groups on lysine residues of proteins to formamide bonds. Alternatively, a targeting molecule and/or a chelator maybe derivatized to expose or attach additional reactive functionalgroups. The derivatization may involve attachment of any of a number oflinker molecules such as those available from Pierce Chemical Company,Rockford, Illinois. (See the Pierce 1986-87 General Catalog, pages313-54.) Alternatively, the derivatization may involve chemicaltreatment of the protein (which may be an antibody). Procedures forgeneration of free sulfhydryl groups on antibodies or antibody fragmentsare also known. (See U.S. Pat. No. 4,659,839.) Maleimide conjugationgroups on a chelator are reactive with the sulfhydryl (thiol) groups.

Alternatively, when the targeting molecule is a carbohydrate orglycoprotein, derivatization may involve chemical treatment of thecarbohydrate; e.g., glycol cleavage of the sugar moiety of aglycoprotein antibody with periodate to generate free aldehyde groups.The free aldehyde groups on the antibody may be reacted with free amineor hydrazine conjugation groups on the chelator to bind the chelatorthereto. (See U.S. Pat. No. 4,671,958.)

The radiolabeled targeting molecules of the present invention have usein diagnostic and therapeutic procedures, both for in vitro assays andfor in vivo medical procedures. The radiolabeled molecules may beadministered intravenously, intraperitoneally, intralymphatically,locally, or by other suitable means, depending on such factors as thetype of target site. The amount to be administered will vary accordingto such factors as the type of radionuclide (e.g., whether it is adiagnostic or therapeutic radionuclide), the route of administration,the type of target site(s), the affinity of the targeting molecule forthe target site of interest, and any cross-reactivity of the targetingmolecule with normal tissues. Appropriate dosages may be established byconventional procedures and a physician skilled in the field to whichthis invention pertains will be able to determine a suitable dosage fora patient. A diagnostically effective dose is generally from about 5 toabout 35 and typically from about 10 to about 30 mCi per 70 kg bodyweight. A therapeutically effective dose is generally from about 20 mCito about 300 mCi. For diagnosis, conventional non-invasive procedures(e.g., gamma cameras) are used to detect the biodistribution of thediagnostic radionuclide, thereby determining the presence or absence ofthe target sites of interest (e.g., tumors).

To render the ester in the conjugates of the present invention moresusceptible to cleavage in the kidneys, an agent that raises urine pHmay also be administered to the patient. Such agents include, forexample, a salt of ascorbate (e.g., sodium ascorbate) or a bicarbonatesalt (e.g., sodium bicarbonate), which may be administeredintravenously. Raising the urine pH to a basic level promotes cleavageof the ester in conjugates or catabolites thereof localized in thekidneys. Clearance of the released radionuclide metal chelates from thebody is thereby enhanced. Administration of such agents to promotecleavage of ester linkers in vivo is described in U.S. patentapplication Ser. No. 07/251,900, which is hereby incorporated byreference.

The comparatively low intestinal localization of the therapeuticradiolabeled antibodies of the present invention or catabolites thereofpermits increased dosages, since intestinal tissues are exposed to lessradiation. The clarity and accuracy of diagnostic images also isimproved by the reduced localization of radiolabeled antibodies orcatabolites thereof in normal tissues.

The following examples are presented to illustrate certain embodimentsof the present invention.

EXAMPLE 1 Synthesis of serine succinate reagent 6

The synthesis procedure is generally as depicted in FIG. 1.

t-butyl, succinimidyl succinate 4 (LG694-73): To an ice cold solution ofsuccinic acid mono-t-butyl ester 3 (870 mg, 5.0 mmol) and NHS (630 mg,5.5 mmol) in acetonitrile (7.0 mL) was added DCC (1130 mg, 5.5 mmol).The reaction was allowed to warm to room temperature and stirred for 4.5hours. The reaction was cooled to 0° C., treated with 0.1 mL aceticacid, and filtered. The filtrate was evaporated to give a gummy solid(1280 mg, theoretical yield). ¹ H NMR (CDCl₃): 1.40 (s, 9H), 2.60 (t,2H), 2.80 (s, 4H), 2.90 (t, 2H).

serinyl succinate 5 (LG694-9.7): To an ice cold suspension of sodiumhydride (60 mg, 2.49 mmol) in DMF (1.0 mL), was added a solution ofN--BOC serine (170 mg, 0.83 mmol). The suspension was stirred for 30minutes and then treated with a solution of 4 (225 mg, 0.83 mmol) in DMF(1.0 mL). The suspension was warmed to room temperature and stirred for16 hours. The reaction was quenched at 0° C. by the addition of asolution of acetic acid (0.1 mL) in EtOAc (1.0 mL). The suspension waspartitioned between EtOAc and pH 4.0 buffer. The aqueous was extractedwith EtOAc (2×30 mL). The aqueous was acidified to pH 1.0 with 1.0 M HCland further extracted with EtOAc (30 mL). The combined EtOAc extractswere washed with brine, dried, and evaporated to give an oil.Chromatography afforded 5 as a colorless oil (190 mg, 0.53 mmol, 53%). ¹H NMR (CDCl₃): 1.40 (2 overlapping singlets, 18H), 2.55 (broad s, 4H),4.40-5.50 (m, 2H), 4.60 (broad s, 1H), 5.50 (broad d, 1H). MS m/e (relintensity): 362 (M+H, 13), 337 (22), 250 (52), 154 (100).

serinyl succinate NHS ester 6 (LG694-79): To an ice cold solution of 5(600 mg, 1.66 mmol) and NHS (229 mg, 1.99 mmol) in acetonitrile (2.5 mL)was added DCC (394 mg, 1.91 mmol). The reaction was warmed to roomtemperature and stirred for 2 hours. The reaction was cooled to 0° C.,treated with acetic acid (0.1 mL), and filtered. The filtrate wasevaporated to give 6 as an oil (760 mg, 1.66 mmol, theoretical yield).1H NMR (CDCl₃): 1.45 (2 overlapping singlets, 18H), 2.55-2.70 (m, 4H),2.85 (s, 4H), 4.55 (dd, 2H), 5.05 (broad s, 1H), 5.60 (broad d, 1H).

EXAMPLE 2 Synthesis of cysteine-serine succinate 13

The procedure is generally as depicted in FIG. 2. S--acm--N--tBOCcysteine TCE ester 7 (JRW 443-14): DCC (794 mg, 3.85 mmol) was added toa solution of S--acm--N--tBOC cysteine (966 mg, 3.50 mmol) andN,N-dimethyl aminopyridine (47 mg, 0.385 mmol) in trichloroethanol (0.37mL, 3.85 mmol) and acetonitrile (18 mL). The suspension was stirred atroom temperature for 48 hours. The suspension was filtered. The filtratewas evaporated. The residue was dissolved in EtOAc (75 mL) and washedwith saturated NaHCO₃ (2×50 mL). The EtOAc was dried, evaporated to givean oil which was crystallized from ether/hexanes to give 7 as a whitesolid (500 mg, 1.22 mmol, 35%). 1H NMR (CDCl₃): 1.50 (s, 9H), 2.05 (s,3H), 3.10 (dd, 2H), 4.40-4.60 (m, 2H), 4.60-4.95 (overlapping dd and s,3H), 5.70 (broad d, 1H), 6.90 (broad s, 1H). M.p. 76°-77° C.cys--ser--succinate 9 (LG 694-80): To an ice cold solution of 7 (772 mg,1.66 mmol) in CH₂ Cl₂ (3.0 mL), was added trifluoroacetic acid (4.0 mL).The reaction was warmed to room temperature and stirred for 1 hour. Thesolution was coevaporated with carbon tetrachloride (3×30 mL). Theresidue (8) was dissolved in DMF (1.5 mL), cooled to 0°, and treatedwith triethylamine (0.24 mL, 1.72 mmol). To this solution was added asolution of 6 (760 mg, 1.66 mmol) in DMF (1.5 mL). To this was addedtriethylamine 0.48 mL, 3.32 mmol). The reaction was warmed to roomtemperature and stirred for 16 hours. The solution was diluted withEtOAc (50 mL) and washed with 0.1M HCl, brine, dried, and evaporated togive an oil (1.4 g). The oil was chromatographed (50% EtOAc:Hexanes 1%HOAc, 700 mL, then 75% EtOAc:Hexanes 1% HOAc, then 99:1 EtOAc:HOAc, 200mL) to give 9 as an oil (200 mg, 0.30 mmol, 18%). ¹ H NMR (CDCl₃): 1.50(overlapping singlets, 18H), 2.05 (s, 3H), 2.55 (broad s, 4H), 3.15 (dd,2H), 4.40-4.65 (m, 5H), 4.70-5.05 (overlapping broad s, dd 3H), 5.70(broad d, 1H), 6.85 (broad s, 1H), 7.75 (broad d, 1H). MS m/e (relintensity): 668 (M+2, 5), 666 (M, 5), 568 (11), 510 (19), 439 (25), 57(100).

cysteine--ser--succ SEOE ma 11(LG694-81): To an ice cold solution of 9(200 mg, 0.30 mmol) in CH₂ Cl₂ (2.0 mL), was added trifluoroacetic acid(2.0 mL). The solution was stirred at room temperature for 1 hour. Thesolution was coevaporated with carbon tetrachloride (3×30 mL). Theresidue was dissolved in DMF (0.5 mL) and cooled to 0°. To this solutionwas added triethylamine (42 μL, 0.30 mmol). To this was addedS-ethoxyethyl mercaptoacetic acid succinimidyl ester 42 (94 mg, 0.36mmol) and triethylamine (84 μL, 0.60 mmol). The ice bath was allowed tomelt and the reaction was stirred at room temperature for 18 hours. Thesolution was diluted with EtOAc (50 mL) and washed with pH 4.0 buffer.The aqueous was extracted with EtOAc (25 mL). The combined EtOAcextracts were washed with brine, dried, and evaporated to give an oil.The oil was chromatographed (23 mm column, 1:1 EtOAc:Hexanes 1% HOAc,400 mL, then 99:1 EtOAc:HOAc, 700 mL, and lastly 10% IPA:EtOAc 1% HOAc)to give 11 as a foam (80 mg, 0.12 mmol, 41%). 1H NMR:1.25 (t, 3H), 1.50(d, 3H), 2.05 (s, 3H), 2.50-2.70 (m, 4H), 3.30 (m, 2H), 3.40-4.30 (m,5H), 4.35-4.95 (m, 8H), 6.90 (broad s, 1H), 7.55-7.65 (broad t, 1H),7.90-8.05 (broad t, 1H). MS m/e: 678 (M+Na), 610, 391, 149.

TFP ester 12 (LG694-82): To an ice cold solution of 11 (55 mg, 0.08mmol) in THF (0.50 mL), were added tetrafluorophenol (18 mg, 0.11 mmol)and DCC (19 mg, 0.10 mmol). The ice bath was removed and the solutionwas stirred at room temperature for 18 hours. The mixture was cooled to0°, treated with acetic acid (0.1 mL), and filtered. The filtrate wasevaporated to give an oil. The oil was chromatographed (1:1EtOAc:Hexanes 1% HOAc, 200 mL, then 99:1 EtOAc:HOAc, 200 mL) to give 12as an oil (45 mg, 0.055 mmol, 70%). ¹ H NMR (CDCl₃): 1.20-1.35 (m, 3H),1.55 (d, 3H), 2.10 (s, 3H), 2.85 (t, 2H), 3.05-3.15 (m, 4H), 3.40 (m,2H), 3.50-3.85 (m, 2H), 4.35-5.05 (m, 9H), 6.70 (broad s, 1H), 6.90-7.15(m, 1H), 6.90-7.15 (m, 1H), 7.70 (m, 1H), 8.20 (broad s, 1H). MS m/e(rel intensity): 806 (M+2, 14), 804 (M, 12), 761 (64), 759 (52), 735(35), 733 (28), 689 (37), 687 (37) 663 (42), 436 (41), 410 (50), 155(100).

cys--ser succinate 13 (LG 694-85): To a solution of 12 (30 mg, 0.037mmol) in THF (0.4 mL) and 1.0M KH₂ PO₄ (80 μL) was added zinc dust (39mg, 0.59 mmol). After 1 hour, additional 1.0 M KH₂ PO₄ (80 μL) and zincdust (39 mg, 0.59 mmol) were added. The mixture was agitated in asonicator for 2 hours. The mixture was filtered, rinsed withacetonitrile and 50% CH₃ CN/H₂ O 1% HOAc. The filtrate was evaporated.The residue was chromatographed (10% IPA:CH₂ Cl₂ 1% HOAc, 50 mL, then25% IPA:CH₂ Cl₂ 2% HOAc) to give 13 as a foam (14 mg, 0.02 mmol, 57%). ¹H NMR (CDCl₃): 1.25 (t, 3H), 1.60 (d, 3H), 2.05 (s, 3H), 2.85 (t, 2H),3.10 (m, 4H), 3.35 (m, 2H), 3.40-3.80 (m, 2H), 4.30-5.00 (m, 9H), 6.65(m, 1H), 6.90-7.10 (m, 1H), 7.70 (m, 1H), 8.20 (broad s, 1H). Analternative and preferred procedure for synthesizing chelating compound13 is presented in example 11.

EXAMPLE 3 Synthesis of N₃ S serine succinate 20

The procedure is generally as depicted in FIG. 3.

N--Cbz--gly--gly TCE ester 14 (LG694-99): To a suspension ofN--Cbz--gly--gly (2.00 g, 7.52 mmol) in acetonitrile (35.0 mL) was addedtrichloroethanol (0.94 mL). To this suspension was added4-dimethylaminopyridine (1.107 g, 9.02 mmol). The solution was stirredfor 10 minutes and then treated with DCC (1.86 g, 9.02 mmol). Themixture was stirred for 18 hours. The mixture was filtered. The filtratewas evaporated. The residue was dissolved in EtOAc (50 mL) and washedwith saturated NaHCO₃ (50 mL). The aqueous was extracted with EtOAc(2×30 mL). The combined EtOAc extracts were washed with brine, dried andevaporated to give an oil. The oil was crystallized from EtOAc/Hexanesto give 14 as a white solid (2.44 g, 6.16 mmol, 82%). ¹ H NMR (CDCl₃):3.95 (d, 2H), 4.20 (d, 2H), 4.80 (s, 2H), 5.15 (s, 2H), 5.50 (broad s,1H), 6.70 (broad s, 1H), 7.35 (s, 5H).

gly--gly TCE ester 15 (LG762-4): A suspension of 14 (1.60 g, 4.0 mmol)in acetic acid (16 mL) was gently heated to complete dissolution of 14.To this solution was added dropwise 30% HBr/HOAc (16 mL). The mixturewas kept at room temperature for 2 hours and then diluted with Et₂ O andevaporated. The residue was evaporated twice from heptane and thendiluted with Et₂ O. The mixture was filtered. The solid was collected byfiltration and dried in vacuo, to give 15 as a white solid (1.37 g, 4.0mmol, theoretical yield). ¹ H NMR (DMSO): 3.65 (broad d, 2H), 4.15 (d,2H), 4.95 (s, 2H), 8.00 (broad s, 1H), 8.90 (broad t, 1H).

N--BOC--T--Butyl ester (LG762-3): To an ice cold solution of 15 (656 mg,1.91 mmol) and 6 (797 mg, 1.74 mmol) in DMF (7.0 mL), was addedtriethylamine (0.36 mL, 2.60 mmol). The ice bath was allowed to melt andthe reaction was stirred at room temperature for 18 hours. The solutionwas evaporated. The residue was dissolved in EtOAc (50 mL) and washedwith pH 4.0 buffer. The aqueous was extracted with EtOAc (2×25 mL). Thecombined EtOAc extracts were washed with brine, dried, and evaporated togive an oil. The oil was chromatographed (1:1 EtOAc:Hexanes 1% HOAc, 120mL, then 99:1 EtOAc:HOAc, 200 mL) to give 16 as a white foam (720 mg,1.19 mmol, 68%). ¹ H NMR (CDCl₃): 1.50 (s, 18H), 2.60 (s, 4H), 4.10 (m,2H), 4.20 (d, 2H), 4.40-4.60 (m, 3H), 4.80 (s, 2H), 5.75 (broad d, 1H),7.20-7.40 (m, 2H).

ser--succinic acid 18 (LG762-5): To an ice cold solution of 16 (360 mg,0.59 mmol) in CH₂ Cl₂ (2.0 mL) was added trifluoroacetic acid (2.0 mL).The reaction was warmed to room temperature and stirred for 1 hour. Thesolution was coevaporated with carbon tetrachloride (3×20 mL). Theresidue, 17, was rinsed with Et₂ O and dried. The residue was dissolvedin DMF (2.0 mL), cooled to 0°, and treated with S-ethoxyethylmercaptoacetic acid succinimidyl ester 42 (186 mg, 0.71 mmol). To thiswas added triethylamine (0.21 mL, 1.48 mmol). The ice bath was allowedto melt and the reaction was stirred at room temperature for 18 hours.The solution was poured into EtOAc (40 mL) and washed with pH 4.0buffer. The aqueous was extracted with EtOAc (30 mL). The aqueous wasacidified to pH 1.0 with 1.0M HCl, and extracted with EtOAc (2×30 mL).The combined EtOAc extracts were washed with brine, dried, andevaporated to give an oil. The oil was chromatographed (99:1 EtOAc:HOAc)to give 18 as a foam (200 mg, 0.33 mmol, 56%). ¹ H NMR (CDCl₃): 1.20(td, 3H), 1.55 (dd, 3H), 2.65 (s, 4H), 3.35 (dd, 2H), 3.45-3.80 (m, 2H),3.90-4.60 (m, 7H), 4.70-4.85 (m, 3H), 7.40-7.70 (m, 3H).

TFP ester 19 (LG762-6): To a solution of 18 (200 mg, 0.33 mmol) andtetrafluorophenol (83 mg, 0.49 mmol) in acetonitrile (1.20 mL) was addedDCC (83 mg, 0.40 mmol). The mixture was stirred at room temperature for18 hours. The mixture was cooled to 0° and filtered. The filtrate wasevaporated. The residue was chromatographed (75% EtOAc¹ Hexanes 1% HOAc)and gave 19 as an oil (150 mg, 0.20 mmol, 61%). ¹ H NMR (CDCl₃): 1.25(td, 3H), 1.55 (dd, 3H), 2.85 (t, 2H), 3.05 (t, 2H), 3.30 (m, 2H),3.40-3.80 (m, 2H), 3.95-4.10 (m, 2H), 4.20 (d, 2H), 4.40-4.90 (m, 6H),6.90-7.20 (m, 2H), 7.20-7.35 (m, 1H), 7.50-7.70 (m, 1H).

N₃ S acid 20 (LG762-7): To a solution of 19 (150 mg, 0.20 mmol) in THF(1.30 mL) and 1.0M KH₂ PO₄ (0.27 mL) was added zinc dust (138 mg, 2.12mmol). The reaction mixture was stirred for 40 minutes. Additional 1.0MKH₂ PO₄ (0.27 mL) and zinc dust (138 mg, 2.12 mmol) were added. Themixture was stirred for 1 hour and then agitated in a sonicator for 25minutes. The mixture was filtered through Celite, rinsed withacetonitrile, and 40% CH₃ CN/H₂ O 1% HOAc. The filtrate was evaporated.The residue was chromatographed (15% IPA:CH₂ Cl₂ 1 % HOAc) and gave 20as a white foam (48 mg, 0.08 mmol, 39%). ¹ H NMR: 1.10 (t,3H), 1.45 (d,2H) 2.70 (t, 2H), 3.05 (t, 2H), 3.20-3.40 (H₂ O in DMSO), 3.60-3.80 (m,3H), 4.10-4.35 (m, 4H), 4.60-4.85 (m, 2H), 7.70-8.10 (m, 1H), 8.20-8.50(m, 2H).

EXAMPLE 4 Preparation of Radionuclide Metal Chelates and Attachment ofthe Chelates to Targeting Proteins

99mTc Chelates: Chelating compounds 13 and 20, synthesized in Examples1-3, were both radiolabeled with ^(99m) Tc according to the followingprocedures:

METHOD A

One mL of sterile water for injection was added to a sterile vialcontaining a stannous gluconate complex (50 mg sodium gluconate and 1.2mg stannous chloride dihydrate, available from Merck Frosst, Canada, indry solid form lyophilized at pH 6.1-6.3) and the vial was gentlyagitated until the contents were dissolved. A sterile insulin syringewas used to inject 0.1 mL of the resulting stannous gluconate solutioninto an empty sterile vial. Sodium pertechnetate (1.0 mL, 75-100 mCi,eluted from a ⁹⁹ Mo/⁹⁹ Tc generator purchased from DuPont, Mediphysics,Mallinckrodt or E. R. Squibb) was added, and the vial was agitatedgently to mix the contents, then incubated at room temperature for 1-2minutes to form a ^(99m) Tc-gluconate complex.

METHOD B

In an alternative procedure for providing the ^(99m) Tc-gluconateexchange complex, the kit includes a vial containing a lyophilizedpreparation at pH 1.8 with sulfuric acid comprising 2.5 mg sodiumgluconate, 0.10 mg stannous chloride dihydrate, 0.1 mg gentisic acid asa stabilizer compound, and about 40 mg lactose as a filler compound toaid in lyophilization. One mL of sodium pertechnetate (about 100 mCi)was added directly to the lyophilized preparation. The vial was agitatedgently to mix the contents to form the ^(99m) Tc-gluconate complex.

A separate vial containing 0.3 mg of a chelating compound in dry solidform was prepared by dispensing a solution of 0.3 mg chelating compound(either compound 13 or 20) in acetonitrile into the vial, then removingthe solvent under N₂ gas. To this vial was then added 0.9 mL of 100%isopropyl alcohol, and the vial was gently shaken for about 2 minutes tocompletely dissolve the chelating compound. Next, 0.6 mL of thissolution of the chellating compound was transferred to a vial containing0.16 mL of glacial acetic acid/0.2N HCl (2:14), and the vial was gentlyagitated. Of this acidified solution, 0.5 mL was transferred to the vialcontaining the ^(99m) Tc-gluconate complex, prepared in Method A above.To the Tc-gluconate complex prepared in Method B, 0.1 mL of thechelating compound in isopropanol (1.0 mg/mL) and 0.4 mL isopropanolwere added. After thorough agitation to mix, the vials from both MethodsA and B were incubated in a 75° C.±2° C. water bath for 15 minutes toform the ^(99m) Tc-chelate, then immediately transferred to a 0° icebath for 2 minutes.

The Fab fragment of a monoclonal antibody (10 mg in 0.5 mL of PBS) wasgenerated by treating the monoclonal antibody with papain according toconventional techniques. The monoclonal antibody, designated NR-LU-10,recognizes a pancarcinoma antigen. Other proteins may be substituted forthe NR-LU-10 Fab fragment.

Either of the vials containing the acidified solution of the ^(99m)Tc-labeled chelate (see above) was removed from the ice bath, 2.0 mL of250 mM sodium bicarbonate buffer pH 9.3 was added, and the vial wasagitated to mix. Immediately, the antibody solution (above) was added,gently agitated to mix and incubated at room temperature for 10 minutesto allow conjugation of the radiolabeled chelate to the antibody.

A column containing an anion exchanger, either DEAE-Sephadex orQAE-Sephadex, was used to purify the conjugate. The column was preparedunder aseptic conditions as follows. Five 1 mL QAE-Sephadex columns wereconnected end-to-end to form a single column. Alternatively, a single 5mL QAE-Sephadex column may be used. The column was washed with 5 mL of37 mM sodium phosphate buffer, pH 6.8. A 1.2μ filter (available fromMillipore) was attached to the column, and a 0.2μ filter was attached tothe 1.2μ filter. A 22-gauge sterile, nonpyrogenic needle was attached tothe 0.2μ filter.

The reaction mixture was drawn up into a 5 mL syringe, and any airbubbles were removed from the solution. After removal of the needle, thesyringe was connected to the QAE-Sephadex column on the end opposite thefilters. The needle cap was removed from the 22-gauge needle attached tothe filter end of the column and the needle tip was inserted into asterile, nonpyrogenic test tube. Slowly, over 2 minutes, the reactionmixture was injected into the column. The eluant was collected into asterile, nonpyrogenic 10 mL serum vial. The now empty syringe on top ofthe column was replaced with a 5 mL syringe containing 5 mL of 150 mM(0.9%) sodium chloride solution (from which air bubbles had beenremoved). Slowly, over 2 minutes, the NaCl solution was injected intothe column, and the eluent was collected in the serum vial. Theradiolabeled antibody fragments were thus recovered from the reactionmixture.

¹⁸⁸ RE CHELATES

The same chelating compounds may be radiolabeled with ¹⁸⁸ Re by aprocedure similar to the ^(99m) Tc labeling procedure. Sodium perrhenateproduced from a W-188/Re-188 research scale generator is combined withcitric acid (a preferred complexing agent for ¹⁸⁸ Re), a reducing agent,and preferably gentisic acid and lactose. The resulting ¹⁸⁸ Re-citrateexchange complex is heated with the desired chelating compound, asabove. A C₁₈ reversed phase low pressure material (Baker C₁₈ acartridges) may be used to purify the ¹⁸⁸ Re-chelate. A monoclonalantibody or fragment thereof is reacted with the chelate in a bufferedsolution to bind the chelate thereto, as described for the ^(99m) Tcprocedure. A Sephadex G-25 column may be used to purify the radiolabeledantibody.

EXAMPLE 5 Preparation of Chelating Compound 28

The procedure is generally as shown in FIG. 4. Butanedioic acidmonobenzylester 21

To a solution of 4.13 mL (4.32 g, 40 mmol) of benzyl alcohol and 4.40 g(44 mmol) of succinic anhydride in 100 mL of THF at 0° C. was added atonce 12.24 mL (8.90 g, 88 mmol) of Et₃ N. The mixture was allowed tocome to room temperature and stirred for 18 h at which time it wasconcentrated under vacuum to a viscous oil and partitioned between 100mL of 1N HCl and 100 mL of EtOAc. The EtOAc layer was extracted with 84mL of 5% NaHCO₃ solution. The aqueous layer was acidified withconcentrated HCl solution to pH 1 and extracted with two 50 mL portionsof EtOAc. The EtOAc layers were combined and washed with brine, dried(M_(g) SO₄), filtered, and concentrated to give 6.9 g (83%) of 21 as awhite chunky solid: ¹ H NMR (CDCl₃) 2.70 (s, 4H), 5.22 (s, 2H), 7.38 (s,5H).

O-(3-Carbobenzoxypropanoyl)-N-Cabobenzoxyserine 22

To a solution of 4.08 g (19.6 mmol) of 21 in 20 mL of CH₂ Cl₂ was added2.14 mL (3.50 g, 29.4 mmol) of SOCl₂. The mixture was stirred at roomtemperature for 6 h and concentrated under vacuum. The resulting yellowoil was dissolved in 20 mL of CH₂ Cl₂, and the solution was cooled to 0°C. To the mixture was added 4.96 g (19.6 mmol) of N-carbobenzoxyserine,3.17 mL (3.10 g, 39.2 mmol) of pyridine, and 244 mg (2.0 mmol) ofN,N-dimethyl-4-aminopyridine. The mixture was allowed to warm to roomtemperature, stirred for 16 h, and partitioned between 100 mL of EtOAcand 100 mL of 1N HCl. The HCl layer was washed with 50 mL of EtOAc andthe combined EtOAc layers were washed with brine, dried (MgSO₄),filtered, and concentrated to give 9.08 g of viscous yellow oil.Purification by chromatography on silica gel (75% EtOAc/25% hexanes/1%HOAc) gave 7.68 g (91%) of 22 as a viscous oil: ¹ H NMR (CDCl₃)2.54-2.71 (m, 4H), 4.30-7.72 (m, 3H), 5.09 (s,2H), 5.11 (s, 2H), 5.78(m, 1H), 7.31 (s, 10H).

O-(3-Carbobenzoxypropanoyl)-N-Carbobenzoxyserine N'-Hydroxysuccinimidylester, 23

To a solution of 1.60 g (3.73 mmol) of 22 in 20 mL of CH₃ CN at 0° C.was added 471 mg (4.09 mmol) of N-hydroxysuccinimde followed by 1.15 g(5.59 mmol) of N,N¹ -dicyclohexylcarbodiimide. The mixture was stirredat 0° for 1 h and placed in the freezer for 16 h. To the mixture wasadded 200 μL of HOAc, and the mixture was placed back in the freezer foran additional 2 h. The solids were removed by filtration, and thefiltrate was concentrated to give a viscous oil containing white solid.Purification by chromatography on silica gel (40% EtOAc/60% hexanes/1%HOAc) gave 1.34 g (68%) of 23 as a viscous oil: ¹ H NMR (CDCl₃) 2.71 (s,4H), 2.80 (s, 4H), 4.40-4.72 (m, 3H) 5.08 (s, 2H), 5.12 (s, 2 H), 5.80(m, 1H), 7.31 (s, 10H).

O-(3-Carbobenzoxypropanoyl)-N-Carbobenzoxyserylolycylolysine O-t-butylester 24

To a suspension of 355 mg (1.58 mmol) of glycylglycine O-t-butyl esterhydrochloride [prepared as described by Moore et al JCS(C) 2349, 1966]in a solution of 756 mg (1.43 mmol) of 23 in 2.8 mL of DMF at 0° wasadded at once 472 μL (4.34 mg, 4.29 mmol) of N-methylmorpholine. Themixture was stirred for 2 h at 0° and partitioned between 10 mL of 1NHCl and 2×20 mL of EtOAc. The combined EtOAc layers were washed with satNaCl, dried (MgSO₄), filtered, and concentrated to give an oil which waspurified by chromatography on silica gel (80% EtOAc/20% hexanes) to give460 mg (54%) of 24 as a sticky gum: ¹ H NMR (DMSO) 1.41 (s, 9H), 2.58(m, 4H), 3.75 (m, 4H), 4.03-4.52 (m, 3H), 5.05 (s, 2H), 5.10 (s, 2H),7.36 (s, 10H), 7.66 (d, 1H), 8.15 (t, 1H), 8.39 (t, 1H).

S-Isobutrylmercaptoacetic acid N-hydroxysuccinimidyl ester 25

To a solution of 61 mg (0.47 mmol) of CoCl₂ in 20 mL of acetonitrileunder nitrogen atmosphere was added dropwise over 15 minutes, a solutionof 4.92 mL (5.0 g, 47 mmol) of isobutryl chloride and 2.97 mL (3.94 g,43 mmol) of mercaptoacetic acid in 50 mL of acetonitrile. The mixturewas stirred at room temperature for 2 hours and concentrated undervacuum. The resulting blue oil was partitioned between 50 mL of 0.1N HCland 100 mL of ether. The ether layer was washed with brine andconcentrated to give an oil. Purification by silica gel chromatography(26% ethyl acetate, 4% acetic acid, 70% hexanes) yielded 3.30 g (47%) ofS-isobutrylmercaptoacetic acid as an oil: ¹ H NMR (CDCl₃): δ 1.23 (d,6H), 2.84 (m, 1H), 3.85 (s, 2H).

To a solution of 3.30 g (20 mmol) of the above acid in 100 mL ofmethylene chloride at 0° C. was added 2.53 g (22 mmol) ofN-hydroxysuccinimide followed by 4.52 g (22 mmol) ofN,N'-dicyclohexylcarbodiimide. The mixture was allowed to stir for 16hours allowing the ice bath to equilibrate to room temperature. Themixture was chilled and filtered through celite. The filtrate wasconcentrated and purified by silica gel chromatography (50% EtOAc-50%hexanes) to give 4.48 g of 25 as a viscous oil: ¹ H NMR (CDCl₃): δ 1.23(d, 6H), 2.80 (m, 1H), 2.81 (s, 4H), 3.98 (s, 2H).

N-(S-Isobutyrylmercaptoacetyl)-(O-3-carboxypropanoyl)-serylglycylglysineO-t-butyl ester 26

A 250 mL hydrogenation flask was charged with 460 mg (0.77 mmol) of 24,7.7 mL of methanol, and 77 mg of 10% palladium on charcoal. The mixturewas shaken under 60 psi of H₂ for 2 h, filtered, and concentrated togive 260 mg of a glassy solid. This material was dissolved in 2.0 mL ofDMF and cooled to 0°, and to the mixture was added 216 mg (0.83 mmol) of25 and 91 μL (84 mg, 0.83 mmol) of N-methylmorpholine. The mixture wasstirred for 2 h at 0°., and 0.5 mL of acetic acid was added. The mixturewas concentrated under vacuum to about 0.5 mL and purified bychromatography on silica gel (96% HOAc/4% HOAc) to give 250 mg of amixture of 26 [¹ H NMR (CDCl₃) 1.21 (d, 6H), 1.48 (s, 9H), 2.55-3.10 (m,5H), 3.61 (m, 2H), 3.95 (m, 4H), 4.70-4.87 (m, 3H), 6.88 (m, 1H), 7.40(m, 2H)] and impurities identified to be N-hydroxysuccinimide, DMF, andacetic acid by NMR. By NMR integration the yield of 26 was determined tobe 151 mg (45%). Compound 26 was used as is in the following step.

N-Hydroxysuccinimidyl Ester of Compound 26, 27

To a solution of 151 mg (0.31 mmol) of 26 and 36 mg (0.31 mmol) ofadditional NHS in 1.5 mL of CH₂ Cl₂ was added 128 mg (0.62 mmol) ofN,N'-dicyclohexylcarbodiimide. The mixture was stirred for 3 h at roomtemperature, 3 drops of acetic acid were added, and the mixture stirredfor an additional 1 h. The mixture was filtered and concentrated to anoil which was purified by silica gel chromatography (7.5/92.5/1,isopropyl alcohol/CH₂ Cl₂ /HOAc) and triturated with Et₂ O to give 120mg (63%) of 27 as a white solid: ¹ H NMR (DMSO) 1.13 (d, 6H), 1.41 (s,9H), 2.68 (t, 2H), 2.82 (s, 4H), 2.93 (t, 2), 2.60-3.0 (buried m, 1H),3.70 (s, 2H), 3.78 (m, 4H), 4.22 (m, 2H), 4.61 (m, 1H), 8.16 (t, 1H),8.46 (m, 2H).

N-(S-Isobutyrylmercaptoacetyl)-O-(3-Carbo-N'-Hydroxysuccinimidylpropanoyl)-Serylolycylglycine28

To a solution of 120 mg (0.19 mmol) of 27 in 750 μL of CH₂ Cl₂ was added750 μL of trifluoroacetic acid. The resulting solution was stirred for1.5 h, concentrated under vacuum, and triturated twice with Et₂ O togive 110 mg of 28 as a white solid. Purification of 30 mg by C₁₈reversed phase HPLC (25% CH₃ CN/75% H₂ /1% HOAc) yielded 13 mg of pure28 which was used for making conjugates: ¹ H NMR (DMSO) 1.12 (d, 6H),2.68 (t, 2H), 2.80 (s, 4H), 2.92 (t, 2H), 2.50-3.00 (buried m, 1H), 3.68(s, 2H), 3.76 (m, 4H), 4.20 (m, 2H), 4.60 (m, 1H), 8.10 (t, 1H), 8.41(m, 2H).

EXAMPLE 6 Synthesis of Chelating Compound 33

The procedure is generally as depicted in FIG. 5.

Compound 22 can be esterified with isobutylene to provide t-butyl ester29 which can be hydrogenated to remove the benzyl ester and the benzylcarbamate protecting groups. Acylation of the deprotected amino groupwith compound 30 (prepared analogously 55 using 25 instead of 42) canprovide 31. Analogously to the previous example, compound can beconverted to compound 33 by forming the NHS ester and removing thet-butyl ester to provide 33.

EXAMPLE 7 Preparation of Protein Conjugate 34

To a solution of 262 μL (5 mg, 1×10⁻⁴ mmol) of 19 mg/mL Fab fragment ofa monoclonal Ab (NRML-05) and 738 μL of pH 7.5 buffer (100 mM sodiumphosphates, 150 mM sodium chloride) was added over 5 seconds with rapidmixing 7.4 μL (0.112 mg, 2×10⁻⁴ mmol) of a 15 mg/mL solution of 28 inDMSO. The mixture was gently mixed to ensure mixing for 1 h, and theconjugated protein 34 was isolated by size exclusion chromatography(PD-10 Sephadex G-25M) eluting with degassed phosphate buffered saline(PBS) to give a 2 mL fraction which contained 3.92 mg of protein asevidenced by UV absorbance at 280 nm. Isoelectric focusing showed ananodal shift indicating that the protein had been modified by increasingthe net negative charge on the protein. Offering larger aliguots ofligand showed larger anodal shifts. The conjugate was concentrated to aconcentration of 4 mg/mL by centrifugal concentration (Centricon-10).

EXAMPLE 8 Deprotection of Conjugate to Provide Free Sulfhydryl ChelatingCompound Conjugate 35

The procedure is generally as depicted in FIG. 6.

To 0.5 mL (2 mg) of a 4 mg/mL solution of 34 in PBS was added 0.5 mL ofdegassed pH 7.6 0.1M sodium phosphate buffer containing 0.5 M hydroxylamine (prepared by dissolving 17.37 g (0.25 mol) of hydroxyl aminehydrochloride and 19.0 g (0.05 mol) of Na₃ PO₄ in 400 mL of water andadjusting to pH 7.6 using 1N NaOH then diluting to 500 mL total volume).The mixture was vortexed and then gently agitated for 15 minutes beforepassing through a size exclusion PD-10 (Sephadex G25n) with degassed PBSto give 2 mL of 35 as a 1 mg/mL protein fraction which was used for Tcchelation.

EXAMPLE 9 Preparation of Radiolabeled Protein 36

The procedure is generally as depicted in FIG. 6.

One mL of sterile water for injection was added to a sterile vialcontaining a stannous gluconate complex (50 mg sodium gluconate and 1.2mg stannous chloride dihydrate, available from Merck Frosst, Canada, indry solid form) and the vial was gently agitated until the contents weredissolved. A sterile insulin syringe was used to inject 0.1 mL of theresulting stannous gluconate solution into an empty sterile vial. Sodiumpertechnetate (0.75 mL, 15-50 mCi/mL, eluted from a ⁹⁹ Mo/⁹⁹ Tcgenerator purchased from DuPont, Mediphysics, Mallinckrodt or E. R.Squibb) was added, and the vial was agitated gently to mix the contents,then incubated at room temperature for 10 minutes to form a ^(99m)Tc-gluconate complex. To 250 μL of a 1 mg/mL solution of 35 in PBS wasadded 87.5 μL of pH 5.5 0.2M acetate buffer and 100 μL of the ^(99m)Tc-gluconate complex. The mixture was placed in a 37° C. incubator for30 minutes and the amount of radionuclide chelated to the conjugate wasdetermined to be 78%. Purification by PD-10 column resulted in a 91%purity with 86% recovery.

EXAMPLE 10 Synthesis of Chelating Compound 47

The procedure is as generally depicted in FIGS. 7-9.

SYNTHESIS OF 37

1,3-Dycyclohexylcarbodiimide (2.10 g, 10.2 mmol) was added to a stirringsolution of N-tert-butoxycarbonyl-L-serine benzyl ester (from BachemInc.) (2.50 g, 8.5 mmol), tert-butyl hydrogen succinate prepared by theprocedures of Buchi, G.; Roberts, E., J. Oro. Chem, 1968, 33, 460 (3,1.48 g, 8.5 mmol) and 4-dimethylaminopyridine (1.14 g, 9.3 mmol) in 17.5mL of anhydrous THF. After stirring at room temperature for 17 hr, themixture was filtered and evaporated in vacuo. The resulting residue wastaken up in EtOAc (30 mL) and washed successively with 1N HCl (2×10 mL),H₂ O (1×10 mL), dried (MgSO₄), filtered, and evaporated. The residue waspurified by flash chromatography on silica gel (30% EtOAc:hexanes). Thepure 37 was isolated as a colorless oil which was used directly in thenext step. ¹ H NMR (CDCl₃) δ 7.39 (br s, 5H), 5.45 (br d, 1H), 5.30 (m,2H), 4.63 (m, 1H) 4.47 (m, 2H), 2.49 (br s, 4H), 1.47 (br s, 18H).

SYNTHESIS OF 38

Compound 37 isolated in the previous step was dissolved in EtOAc (20 mL)and added to 5% palladium on activated carbon (pre-moistened with EtOAc)and shaken under H₂ (60 psi) in a Parr apparatus at 25° C. for 20 h. Thecrude reaction mixture was filtered through Celite, and evaporated invacuo. The residue was purified by flash chromatography on silica gel(50:46:4 EtOAc:hexanes:HOAc) to yield 2.92 g of 38 (95% fromN-tert-butoxy-carbonyl-L-serine benzyl ester). ¹ H NMR (CDCl₃) δ 5.51(br d, 1H), 4.64 (m, 1H), 4.50 (m, 2H), 2.59 (br s, 4H), 1.48 (br s,18H).

SYNTHESIS OF 39

1,3-Dicyclohexylcarbodiimide (1.77 g, 8.57 mmol) was added to a stirringsolution of (2.58 g, 7.14 mmol), 2,2,2-trichloroethanol (0.69 mL, 7.14mmol) and 4-dimethylaminopyridine (1.05 g, 8.57 mmol) in 9.0 mL ofanhydrous THF. After stirring at room temperature for 16 h the mixturewas filtered and evaporated in vacuo. The resulting residue was taken upin EtOAc (50 mL) and washed with 0.1N HCl (3×30 mL), then saturated NaCl(1×20 mL), then dried (MgSO₄), filtered, and evaporated. The residue waspurified by flash chromatography on silica gel (20% EtOAc:hexanes) toyield 1.22 g of 39 (35%). ¹ H NMR (CDCl₃) δ 5.48 (br d, 1H), 4.81 (m,3H), 4.52 (m, 2H), 2.53 (br s, 4H), 1.44 (br s, 18H).

SYNTHESIS OF 40

Compound 39 (0.105 g, 0.213 mmol) was dissolved in trifluoroacetic acid(2.0 mL, 26 mmol) and stirred at room temperature for 1.5 h. The mixturewas evaporated in vacuo to yield 89 mg (93%) of 40 as a colorless oil. ¹H NMR (DMSO) δ 5.08 (m, 2H), 4.77 (m, 1H), 4.45 (d, 2H), 2.51 (m, 4H).

S-(1-Ethoxyethyl)Mercaptoacetic Acid (5a) 41

A solution of mercaptoacetic acid (17.4 mL, 250 mmol) in 125 mL ofdichloromethane containing p-toluenesulfonic acid monohydrate (0.24 g,1.26 mmol) was cooled to -18° to -25° C. with stirring. Ethyl vinylether (23.9 mL, 250 mmol) in 125 mL of dichloromethane was addeddropwise to the cold solution over a period of 90 minutes. The stirringwas continued for an additional 30 minutes with the temperaturemaintained in the -18° to -25° C. range. Then 200 mL of pH=7 phosphatebuffer was added, and the reaction mixture was allowed to warm withstirring for 10 to 15 minutes. The mixture was then poured into a flaskcontaining 900 mL of ethyl acetate and 200 mL of water. Layers wereseparated and the aqueous portion extracted twice with ethyl acetate.The organic layers were combined, washed with brine and dried (MgSO₄).Removal of the solvent left 31.4 g of S-(1-ethoxyethyl)mercaptoaceticacid 41 as a colorless oil (77% yield): ¹ H NMR (CDCl₃) 1.15 (t,J=7.0Hz, 3H), 1.52 (d, J=6.4Hz, 3H), 3.36 (s, 2H), 3.60 (m, 2H), 4.84(q, J=6.4Hz, 1H), 11.65 (s, 1H). The material was used without furtherpurification.

Succinimidyl S-(1-Ethoxyethyl)Mercaptoacetate 42

A solution of S-(1-ethoxyethyl)mercaptoacetic acid (5.76 g, 35.1 mmol)and N-hydroxysuccinimide (4.85 g, 42.1 mmol) was prepared in 100 mL ofanhydrous THF. To this was added a solution of1,3-dicyclohexylcarbodiimide (8.70 g, 42.1 mmol) in 65 mL of anhydrousTHF. The mixture was stirred at room temperature for 2 hours or untilTLC analysis indicated complete formation of the succinimidyl ester. Themixture was then filtered, and the filtrate was concentrated in vacuo toa viscous residue. The residue was dissolved in ethyl acetate, washedwith water, brine, and dried (MgSO₄). Removal of the solvent left thecrude succinimidyl ester as an oil, which was further purified by flashchromatography on silica gel, using ethyl acetate-hexanes as the columneluent, to give 5.1 g of S-(1-ethoxyethyl)mercaptoacetic acidsuccinimidyl ester as a colorless oil (56% yield): ¹ H NMR (CDCl₃) 1.21(t, J=7.0Hz, 3H), 1.58 (d, J=6.4Hz, 3H), 2.83 (s, 4H), 3.60 (m, 4H),4.88 (q, J=6.4Hz, 1H).

SYNTHESIS OF 43

Solid NaHCO₃ (1.09 g, 13.0 mmol) was added to a solution ofglycylglycine (1.22 g, 9.3 mmol) in 10 mL of water. After gas evolutionceased, a solution of 42 (2.66 g, 10.2 mmol) in 12 mL of CH₃ CN wasadded to the reaction mixture. The mixture was stirred at roomtemperature for 22 h, then evaporated in vacuo. The residue was purifiedby flash chromatography on silica gel (85:10:5 CH₃ CN:H₂ O:HOAc) toyield 2.2 g (86%) of 43 as a viscous oil. ¹ H NMR (DMSO) 8.26 (t, 1H),8.08 (t, 1H), 4.80 (q, 1H), 3.73 (m, 4H), 3.52 (m, 2H), 3.24 (s, 2H),1.43 (d, 3H), 1.10 (t, 3H).

SYNTHESIS OF 44

1,3-Dicyclohexylcarbodiimide (0.66 g, 3.2 mmol) was added to a stirringsolution of 43 (0.81 g, 2.9 mmol) and N-hydroxysuccinimide (0.37 g, 3.2mmol) in 10 mL of CH₃ CN. After stirring for 2 h, the mixture wasfiltered and the filtrate was evaporated in vacuo. The residue waspurified by flash chromatography on silica gel (96:4 EtOAc:HOAc) toyield 0.80 g (73%) of 44 as a viscous oil. ¹ H NMR (DMSO) δ 8.54 (t,1H), 8.29 (t, 1H), 4.80 (q, 1H), 4.27 (d, 2H), 3.78 (d, 2H), 3.53 (m,2H), 3.24 (s, 2H), 2.81 (s, 4H), 1.43 (d, 3H), 1.09 (t, 3H).

SYNTHESIS OF 45

Triethylamine (0.039 mL, 0.28 mmol) was added to a solution of 40 (83mgs, 0.18 mmol) and 44 (104 mgs, 0.28 mmol) in 1.0 mL of anhydrous DMF.After stirring at room temperature for 2.5 h the mixture was evaporatedin vacuo. The resulting residue was taken up in EtOAc (20 mL) and washedwith water (5×10 mL), saturated NaCl (1×10 mL), then dried (Na₂ SO₄),filtered, and evaporated. The residue was purified by flashchromatography on Baker C-18 gel (J. T. Baker C₁₈ gel #7025) (50:47:3CH₃ CN:H₂ O:HOAc) to yield 68 mg (62%) of 45 as an oil. ¹ H NMR (DMSO) δ8.58 (d, 1H), 8.29 (t, 1H), 8.20 (t, 1H), 4.93 (m, 2H), 4.79 (m, 2H),4.33 (m, 2H), 3.82 (d, 2 H), 3.73 (d, 2H), 3.52 (m, 2H), 3.22 (s, 2H),2.49 (m, 4H), 1.43 (d, 3H), 1.09 (t, 3H).

SYNTHESIS OF 46

1,3-Dicyclohexylcarbodiimide (37 mg, 0.18 mmol) was added to a stirringsolution of 45 (64 mg, 0.11 mmol) and 2,3,5,6-tetrafluorophenol (46 mg,0.28 mmol) in CH₂ Cl₂ (0.8 mL). After stirring at room temperature for4.5 h, the mixture was filtered and evaporated in vacuo. The residue waspurified by flash chromatography on silica gel (96:4 EtOAc:HOAc) toyield 64 mgs (80%) of 46 as a viscous oil. ¹ H NMR (DMSO) δ 8.59 (d,1H), 8.24 (t, 1H), 8.17 (t, 1H), 7.94 (m, 1H), 4.92 (m, 2H), 4.81 (m,2H), 4.36 (m, 2H), 3.80 (d, 2H), 3.73 (d, 2H), 3.53 (m, 2H), 3.22 (s,2H), 3.04 (m, 2H), 2.76 (m, 2H), 1.42 (d, 3H), 1.08 (t, 3H).

SYNTHESIS OF 47

Zinc dust (124 mg, 1.9 mmol) was added in portions to a stirringsolution of 46 (57 mg, 0.076 mmol) and 1.0M KH₂ PO₄ (0.25 mL) in 0.6 mLof THF. After stirring at room temperature for 1.5 h the mixture wasfiltered through celite and evaporated in vacuo. The residue waspurified by flash chromatography on Baker C-18 gel (50:47:3 CH₃ CN:H₂:HOAc), followed by preparative C-18 HPLC (50:49:1 CH₃ CN:H₂ O:HOAc) toyield 16 mg (34%) of pure 47 as a white solid. ¹ H NMR (DMSO) δ 8.30 (d,1H), 8.26 (t, 1H), 8.17 (t, 1H), 7.96 (m, 1H), 4.81 (q, 1H), 4.59 (m,1H), 4.30 (m, 2H), 3.76 (m, 4H), 3.52 (m, 2H), 3.24 (s, 2H), 3.07 (m,2H), 2.74 (m, 2H), 1.43 (d, 3H), 1.10 (t, 3H).

Chelating compound 47 was radiolabeled and attached to a targetingprotein using the same procedures described in example 4.

EXAMPLE 11 Alternative Route to Compound 9, an Intermediate in theSynthesis of Chelating Compound 13

The synthesis procedure is generally as depicted in FIG. 10.

Synthesis of N-T-BOC-Serine-O-T-Butyldimethylsilyl Ether 48. (LG762-21)

To a solution of N-BOC-serine (615 mg, 3.00 mmol) and imidazole (449 mg,6.60 mmol) in DMF (10.0 mL), was added t-butyldimethylsilyl chloride(994 mg, 6.60 mmol). The reaction solution was stirred at roomtemperature for 15 hours and then concentrated. The residue wasdissolved in EtOAc (50 mL) and washed with water (25 mL), brine (25 mL),and dried to give 48 as a white foam (1.00 g, 3.0 mmol, theoreticalyield). ¹ H NMR (DMSO): 0.05 (s, 4H), 0.90 (s, 9H), 1.40 (s, 9H), 3.80(t, 2H), 4.10 (m, 1H), 6.75 (m, 1H).

Synthesis of N-T-BOC-Serine-O-T-Butyldimethylsilylsuccinimidyl Ester 49(LG762-22)

To an ice cold solution of 48 (1.00 g, 3.0 mmol) in acetonitrile (4.5mL) was added NHS (414 mg, 3.60 mmol) followed by DCC (712 mg, 3.45mmol). The solution was warmed to room temperature and stirred for 16hours The mixture was treated with acetic acid (0.10 mL), cooled to 0°,and filtered. The filtrate was evaporated to give 49 as a white foam(1.25 g, 3.0 mmol, theoretical yield). ¹ H NMR (DMSO): 0.05 (s, 4H),0.90 (s, 9H), 1.40 (s, 9H), 3.80 (s, 4H), 3.90 (m, 2H), 4.50 (m, 1H),7.45 (d, 1H).

SYNTHESIS OF 50 (LG762-32)

To a solution of 7 (1.02 g, 2.41 mmol) in CH₂ Cl₂ (5.6 mL) was addedtrifluoroacetic acid (5.6 mL). The solution was stirred at roomtemperature for 1 hour and then coevaporated with CCl₄ (3×30 mL). Theresidue (8) was dissolved in DMF. To this solution at 0° was added asolution of 49 (1.00 g, 2.41 mmol) in DMF (5.0 mL). To this was addedtriethylamine (0.84 mL, 6.02 mmol). The reaction solution was stirred atroom temperature for 16 hours. Additional triethylamine (0.20 mL) wasadded and the reaction was stirred for 1 hour. The solution wasconcentrated. The residue was dissolved in EtOAc (40 mL) and washed withpH 4.0 buffer. The aqueous was washed with EtOAc (30 mL). The combinedEtOAc extracts were washed with brine, dried, and evaporated to give anoil. The oil was chromatographed (1:1 EtOAc:Hexanes 1% HOAc) to give 50as a white foam (0.89 g, 1.52 mmol, 63%). ¹ H NMR (DMSO): 0.05 (s, 5H),0.80 (s, 9H), 1.35 (s, 9H), 1.85 (s, 3H), 2.95 (ddd, 2H), 3.70 (m, 2H),4.20 (m, 3H), 4.60 (m, 1H), 4.85 (dd, 2H), 6.65 (broad d, 1H), 8.55 (m,2H).

SYNTHESIS OF 51 (LG762-28)

A solution of 50 (200 mg, 0.34 mmol) in 3:1:1 HOAc:H₂ O:THF (1.3 mL) wasstirred at room temperature for 60 hours. The solution was partitionedbetween EtOAc and water. The aqueous was extracted with EtOAc (2×20 mL).The combined EtOAc extracts were washed with brine, dried, andevaporated to give 51 as an oil (175 mg, 0.34 mmol, theoretical yield).¹ H NMR (CDCl₃): 1.40 (s, 9H), 1.95 (s, 3H), 3.05 (ddd, 2H), 3.65 (dd,1H), 4.05 (dd, 1H), 4.15-4.50 (m, 3H), 4.70 (dd, 2H), 4.90 (m, 1H), 5.75(m, 1H), 6.70 (m, 1H), 7.75 (m, 1H).

SYNTHESIS OF 52 (LG762-30)

To an ice cold solution of 51 (90 mg, 0.18 mmol), 3 (31 mg, 0.18 mmol),and DMAP (24 mg, 0.19 mmol) in THF (0.6 mL), was added DCC (43 mg, 0.21mmol). The solution was warmed to room temperature and stirred for 18hours. The mixture was filtered and rinsed with cold acetonitrile. Thefiltrate was evaporated. The residue was chromatographed (50%EtOAc:Hexanes 1% HOAc, 300 mL, then 75% EtOAc:Hexanes 1% HOAc, 300 mL)to give 52 as an oil (60 mg, 0.09 mmol, 51%). ¹ H NMR (CDCl₃): 1.50 (s,18H), 2.05 (s, 3H), 2.60 (broad s, 4H), 3.10 (m, 2H), 4.35-4.60 (m, 5H),4.60-5.05 (overlapping dd and s, 3H), 5.70 (m, 1H), 6.75 (broad s, 1H),7.70 (broad d, 1H).

EXAMPLE 12 Analysis of Stereochemical Isomers of ^(99m) Tc-LabeledChelates

Analyses of the technetium labeled N₃ S and N₂ S₂ chelates wereperformed by isocratic reverse phase C₁₈ HPLC in 24% acetonitrile mobilephase at a flow rate of 1 mL/min. Compound 20 afforded two epimerictechnetium complexes A and B in the ratio of 1:7. Radiometric peaks Aand B refer to the order of elution off HPLC. Compound 47 on the otherhand afforded two diastereometric technetium complexes (Peaks A and B)in the ratio of 1:1. Compound 13 upon technetium complexation resultedin a mixture of epimeric peaks A and B in the ratio of 1:8.

EXAMPLE 13 Biodistribution Studies

Biodistribution of ^(99m) Tc-labeled antibody fragments prepared abovewas analyzed in a rat model. The antibody fragments were labeled with^(99m) Tc chelates prepared from compounds 13, 20, or 47. (See examples4 and 10.)

The three studies were conducted in male Sprague Dawley rats weighingabout 200-300 grams. The rats were received and acclimated to thefacility 5 to 7 days prior to use in a study. Four rats were used foreach timepoint in each study.

The rats were placed under a heat lamp to dilate their tail veins. 100∥g of one of the radiolabeled antibody fragments (in a volume of 200-300μL) was injected into each rat intravenously via the tail vein.Depending on the specific activity of the individual preparation ofradiolabeled antibody fragment, the amount of radioactivity injected wasbetween 200 uCi and 1 mCi. Biodistribution was analyzed at each of fourtimepoints (3 hours, 6 hours, 10 hours, and 20 hours post-injection.) Ateach timepoint, four rats were anesthetized with halothane (inhalant)until they showed no reflexive responses. Three mL of blood was thencollected, followed by sacrifice of the rats using euthanasia solution.Both procedures were done via cardiac puncture.

Each rat was dissected and the following tissues were collected,weighed, and placed in tubes for counting using a gamma counter. Theresults of the three studies are presented in FIGS. 11-13, where thepercentage of the total injected dose of radioactivity that hadlocalized in each of the tissues (the entire organ) is shown. In thefigures, BL represents blood, LV represents liver, ST representsstomach, KD represents kidneys, and 1NT represents intestines. For eachconjugate, Ab represents the monoclonal antibody fragment.

The top graph in each figure presents biodistribution data for the sameantibody fragment labeled with the ^(99m) Tc-N₃ S chelate shown. Thisconjugate does not have a linkage comprising a cleavable ester, and ispresented for comparative purposes.

EXAMPLE 14 Biodistribution Studies for Conjugates Comprising Esters inthe Opposite Orientation

Biodistribution studies were conducted using the procedures of example13 for the following conjugates, in which Ab represents a Fab fragmentof monoclonal antibody NR-LU-10: ##STR24## The first chelate is the sameone used for comparative purposes in example 13. There is no ester inthe linkage between the chelate core and the antibody fragment in theconjugates comprising this chelate. The second chelate comprises anester in the linkage to the antibody fragment, but the ester has anorientation opposite to that of the compounds of the present invention.

The results are presented in FIG. 14. Localization of radioactivitywithin the intestines was relatively high for the second conjugate.Thus, the presence of an ester per se in the linkage was not sufficientto reduce intestinal radioactivity levels over those of the firstconjugate. While not wishing to be bound by theory, the particularester-containing linkage in the second conjugate may not be readilycleavable under physiological conditions. The free acid form of thechelate that would be released by ester cleavage was not detected byHPLC. The relatively high intestinal radioactivity levels may beattributable to hepatobiliary excretion of the lysine adduct of thechelate, which would be expected to be a major catabolite if the esteris not efficiently cleaved.

Another biodistribution study was conducted by the same procedures forthe following conjugates, in which Ab represents the NR-LU-10 Fabantibody fragment: ##STR25##

The results are presented in FIG. 15. As before, the presence of anester (in the orientation opposite to that of the esters in conjugatesof the present invention) in the linkage to the antibody fragment wasnot sufficient to promote enhanced biodistribution properties.Specifically, intestinal radioactivity levels were relatively high forthe ester-containing conjugate "C".

What is claimed is:
 1. A compound of the formula: ##STR26## wherein: mis 0 or 1;R represents H or CH_(3;) X represents O or S; each R₃ isindependently selected from H, CH₂ OH, CH₃, and --(CH₂)_(n) --COOH,wherein n is 0-2, with at least one R₃ substituent being --(CH₂)_(n)--COOH; T' and T each represent hydrogen or a sulfur protecting group;R₂ represents a spacer; and P' represents a targeting molecule or aconjugation group.
 2. The compound of claim 1, wherein R₂ is selectedfrom: --(CH₂)_(n') --, wherein n' is 2-5; ##STR27## wherein W representsan electron withdrawing or electron donating group and m' is 1-4;##STR28## wherein W represents an optional electron withdrawing orelectron donating group.
 3. The compound of claim 2 wherein X is O andR₂ is --(CH₂)₂ --.
 4. A compound of the formula: ##STR29## wherein P'represents a targeting molecule or conjugation group and T and T' eachrepresent a sulfur protecting group.
 5. The compound of claim 4 whereinP' represents an active ester, T represents a hemithioacetal sulfurprotecting group and T' represents an acetamidomethyl group.
 6. Acompound of the formula: ##STR30## wherein p is 0 or 1; one of R' and R"represents ##STR31## and the other is selected from H, CH₃, CH₂ OH,(CH₂)_(n) --CONH₂ and (CH₂)_(n) --COOH wherein n=0-2;m is 0 or 1; R₁represents H or CH₃ ; X represents O or S; T represents hydrogen or asulfur protecting group; R₂ represents a spacer; and P' represents atargeting molecule or a conjugation group.
 7. The compound of claim 6,wherein R₂ is selected from: --(CH₂)_(n') --, wherein n' is 2-5;##STR32## wherein W represents an electron donating or electronwithdrawing group and m' is 1-4; ##STR33## where W represents anoptional electron donating or electron withdrawing group.
 8. Thecompound of claim 6 wherein p is 0, X is 0 and R₂ is --(CH₂)₂ --.
 9. Acompound of the formula: ##STR34## wherein T represents a sulfurprotecting group and P' represents a targeting molecule or a conjugationgroup.
 10. A compound of the formula: ##STR35## wherein T represents asulfur protecting group and P' represents a targeting molecule or aconjugation group.
 11. The compound of claim 9 or 10 wherein T, whentaken together with the sulfur atom attached thereto, represents ahemithioacetal or S-isobutyryl group and P represents an active ester.12. A compound of the formula: ##STR36## wherein p is 0 or 1; each R₄ isindependently selected from H, CH₃, CH₂ OH, (CH₂)_(n) --COHN₂ and(CH₂)_(n) --COOH wherein n=0-2;m is 0 or 1; represents H or CH₃ ; Xrepresents O or S; T represents hydrogen or a sulfur protecting group;R₂ represents a spacer; and P' represents a targeting molecule or aconjugation group.
 13. The compound of claim 12, wherein R₂ is selectedfrom: --(CH₂)_(n) '--, wherein n' is 2-5; ##STR37## wherein W representsan electron donating or donating group and m' is 1-4; ##STR38## whereinW represents an optional electron donating or electron withdrawinggroup.
 14. The compound of claim 12 wherein each R₄ is H, p is 0, X is Oand R₂ is --(CH₂)₁ --.
 15. A compound of the formula: ##STR39## whereinT represents a sulfur protecting group and P' represents a targetingmolecule or a conjugation group.
 16. The compound of claim 15, whereinT, when taken together with the sulfur atom attached thereto, representsa hemithioacetal or S-isobutyryl group and P' represents an activeester.
 17. The compound of claim 1, 6, or 12 wherein the conjugationgroup is selected from the group consisting of active esters,isothiocyanates, amines, hydrazines, thiols, maleimides or other Michaelacceptors, and activated halides.
 18. The compound of claim 17 whereinthe conjugation group is an active ester.
 19. The compound of claim 1, 6or 12 wherein the targeting molecule is a monoclonal antibody or anantigen binding fragment thereof.
 20. A compound of the formula:##STR40## and stereochemical isomers thereof, wherein: M represents aradionuclide metal or oxide thereof;m is 0 or 1; R₁ represents H or CH₃; X represents O or S; each R₃ is independently selected from H, CH₃,CH₂ OH, and --(CH₂)_(n) --COOH, wherein n is 0-2, with at least one R₃substituent being --(CH₂)_(n) --COOH; R₂ represents a spacer; and P'represents a targeting molecule or a conjugation group.
 21. The compoundof claim 20, wherein R₂ is selected from: --(CH₂)_(n') --, wherein n' is2-5; ##STR41## wherein W represents an electron donating or electronwithdrawing group and m' is 1-4; ##STR42## wherein W represents anoptional electron donating or electron withdrawing group.
 22. Thecompound of claim 20 wherein X is O, and R₂ is --(CH₂)₂ --.
 23. Acompound of the formula: ##STR43## and stereochemical isomers thereof,wherein P' represents a targeting molecule or a conjugation group and Mrepresents a radionuclide metal selected from ^(99m) Tc, ¹⁸⁸ Re, and ¹⁸⁶Re.
 24. The compound of claim 23 wherein P' represents an active ester.25. A compound of the formula: ##STR44## and stereochemical isomersthereof, wherein M represents a radionuclide metal or oxide thereof;p is0 or 1; one of R' and R" represents ##STR45## and the other is selectedfrom H, CH₃, CH₂ OH, (CH₂)_(n) --CONH₂ and (CH₂)_(n) --COOH whereinn=0-2; m is 0 or 1; R represents H or CH₃ ; X represents O or S; R₂represents a spacer; and P' represents a targeting molecule or aconjugation group.
 26. The compound of claim 25, wherein R₂ is selectedfrom:--(CH₂)_(n) '--, wherein n' is 2-5; ##STR46## wherein W representsan electron donating or electron withdrawing group and m' is 1-4;##STR47## wherein W represents an optional electron donating orwithdrawing groups.
 27. The compound of claim 25 wherein p is 0, X is O,and R₂ is --(CH₂)₂ --.
 28. A compound of the formula: ##STR48## andstereochemical isomers thereof, wherein M represents a radionuclidemetal selected from ^(99m) Tc, ¹⁸⁸ Re, and ¹⁸⁶ Re, and P' represents atargeting molecule or a conjugation group.
 29. A compound of theformula: ##STR49## and stereochemical isomers thereof, wherein Mrepresents a radionuclide metal selected from ^(99m) Tc, ¹⁸⁸ Re, and ¹⁸⁶Re, and P' represents a targeting molecule or a conjugation group.
 30. Acompound of the formula: ##STR50## and stereochemical isomers thereof,wherein M represents a radionuclide metal or oxide thereof;p is 0 or 1;each R₄ is independently selected from H, CH₃, CH₂ OH (CH₂)_(n) --CONH₂and --(CH₂)_(n) --COOH wherein n=0 or 1; m is 0 or 1; R₁ represents H orCH₃ ; X represents O or S; R₂ represents a spacer; and P' represents atargeting molecule or a conjugation group.
 31. The compound of claim 30,wherein R₂ is selected from:--(CH₂)_(n') --, wherein n' is 2-5;##STR51## wherein W represents an electron donating or electronwithdrawing group and m' is 1-4; ##STR52## wherein W represents anoptional electron donating or withdrawing groups.
 32. The compound ofclaim 30 wherein each R₄ is H, p is 0, X is O, and R₂ is --(CH₂)₂ --.33. A compound of the formula: ##STR53## and stereochemical isomersthereof, wherein M represents a radionuclide metal selected from ^(99m)Tc, ¹⁸⁸ Re, and ¹⁸⁶ Re, and P' represents a targeting molecule or aconjugation group.
 34. The compound of claim 20, 25, or 30 wherein theconjugation group is selected from the group consisting of activeesters, isothiocyanates, amines, hydrazines, thiols, maleimides or otherMichael acceptors, and activated halides.
 35. The compound of claim 34wherein the conjugation group is an active ester.
 36. The compound ofclaim 20, 25 or 30 wherein the targeting molecule is a nonoclonalantibody or an antigen binding fragment thereof.
 37. A method forradiolabeling a targeting molecule, comprising reacting a compound ofclaim 20, 25, or 30, wherein P' represents a conjugation group, with atargeting molecule, thereby attaching said compound to said targetingmolecule.
 38. The method of claim 37, wherein said targeting molecule isa monoclonal antibody or an antigen binding fragment thereof.
 39. Amethod for radiolabeling a targeting molecule, comprising reacting acompound of claim 1, 6, or 12 wherein P' represents a conjugation group,with a targeting molecule, thereby attaching the compound to saidtargeting molecule, and then radiolabeling the compound.
 40. The methodof claim 39 wherein said targeting molecular is a monoclonal antibody oran antigen binding fragment thereof.